In Vitro Gastrointestinal Digestion Affects the Bioaccessibility of Bioactive Compounds in Hibiscus sabdariffa Beverages

Hibiscus sabdariffa possess great versatility to be used as an ingredient for a whole range of products with natural-based ingredients, which are growing in popularity due to the health benefits of bioactive compounds (BC). Therefore, the objective of this study was to characterize the BC content in Hibiscus beverages and to evaluate their in vitro bioaccessibility. Results showed significant differences (p < 0.05) in the total contents of BC prior to the in vitro intestinal digestion. Hibiscus acid was the most abundant compound identified. Thirty-five compounds were identified in the Hibiscus beverage at the initial stage, while a maximum of 15 compounds were quantified in the different fractions of gastrointestinal digestion. After digestion, significant differences were found compared with the initial content of BC. That phenolic acids were the less bioaccessible group, while flavonoids were the most diverse. Principal components analysis showed different clusters and changes in the profiles of BC present at the initial stage and those bioaccessible, showing that intestinal digestion significantly affects the BC profile of the beverage.


Introduction
The study of bioactive compounds (BC) in a wide range of food matrices, their functionality, and their beneficial properties have become an elementary aspect of scientific research and innovation in food products. Beverages that include natural-based compounds are growing in popularity due to the enhanced health benefits provided by these ingredients [1]. Most of the time, consumers are unwilling to compromise taste in exchange for health benefits. Therefore, developing new products that effectively bind consumer expectations and preferences is the current challenge for the food and beverage industries.
In Mexico, Hibiscus calyces are mainly used to prepare a refreshing beverage widely consumed by the population, which gives the H. sabdariffa crop an economical and cultural importance [2,3]. Hibiscus stands out for its characteristic flavor, as well as its potentially beneficial properties to health. It is an essential source of BC, mainly phenolic acids, flavonoids, and anthocyanins [4]. It has been previously reported to have different properties such as diuretic, digestive, anti-inflammatory, and laxative, as well as to moderate hypertension and cholesterol [5][6][7]. However, these compounds must be released during intestinal digestion due to hydrolytic enzymatic activity to be bioaccessible and potentially promote health benefits [8].
Generally, research around H. sabdariffa beverages has approached enhancing the phytochemical and physicochemical composition of calyces extracts or optimizing sensory characteristics and consumer acceptance [9][10][11]. Thus, knowledge of the in vitro bioaccessibility of BC after the consumption of Hibiscus beverages is currently scarce, and the health effects still need to be fully understood. Within the different varieties of H. sabdariffa, an

Initial Bioactive Compounds in Hibiscus Beverages
Retention time (R T ), molecular formula, and accurate mass of the quasimolecular ion [M-H] − [M] + after negative and positive ionization by HPLC-ESI-MS in commercial and Hibiscus beverage at initial content and after the in vitro gastrointestinal digestion are shown in Table 1. The number assigned to the identified compounds was 1-35, depending on R T and group. Regarding organic acids, trimethylhydroxycitric acid I, hibiscus acid dimethylesther, hydroxycitric acid, hibiscus acid, and trimethylhydroxycitric acid II led this group (IDs: 2-6). Concerning hydroxycinnamic acids and derivatives, ten compounds belong to this group such as caffeoylquinic acid, three isomers of coumaroylquinic acid and caffeoylshikimic acid, chlorogenic acid, and caffeic acid (ID: 8-11 and 13-17) while one hydroxybenzoic acid was identified (ID: 12). Regarding flavonoids, 18 compounds were identified, headed by glycosidic derivatives (ID: [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32]. The presence of compounds derived from flavonoids, such as anthocyanins and anthocyanidins, specifically delphinidin-3-sambubioside, was also found, cyanidin-3-glucoside and delphinidin (ID: [33][34][35]. The initial content analysis of the extracts revealed the presence of 35 compounds in the Hibiscus beverage and 11 compounds in the commercial beverage (Table 2). It is outstanding that the content of BC in Hibiscus beverage was three times higher than commercial beverage (417 vs. 138 mg/100 mL). The presence of organic acids was notable. Hibiscus acid was the most abundant compound identified in both samples. Nevertheless, significant differences (p < 0.05) were observed between the samples. The organic acids found in the Hibiscus beverage were significantly higher in concentration compared to those in the commercial beverage, as shown in Table 2. In the commercial beverage, hibiscus acid accounted 70.2 mg/100 mL, while for the Hibiscus beverage had 231.52 mg/100 mL. Since hibiscus acid is the predominant compound in the commercial beverage, this translated into a higher acidity than the Hibiscus beverage, which had repercussions in consumers' acceptance.
Regarding hydroxycinnamic acids and their derivatives, caffeoylquinic acids and isomers of coumaroylquinic and caffeoylshikimic acids were predominant in Hibiscus beverage (up to 51.38 mg/100 mL). In contrast, only caffeoylquinic acid, coumaroylquinic acid II, and chlorogenic acid were detected in minor amounts in the commercial beverage (7.84 mg/100 mL). As previously reported, H. sabdariffa has been considered a rich source of hydroxycinnamic acids and derivatives [4,5] and considering that the Hibiscus beverage contains mint, Eftekhari, et al. [20] reported that hydroxycinnamic acids and its derivatives are commonly found in the genus Mentha. Values represent mean ± SD (n = 3). Different lowercase letters in the same row indicate significant differences between beverages (p < 0.05). n.d. = Not detected.
Up to 18 flavonoids were quantified at the initial stage in the Hibiscus beverage, whereas in contrast, only three flavonoids were identified in the commercial beverage ( Table 2). Glycosidic derivatives of flavonols (quercetin and kaempferol), aglycones (luteolin, quercetin, myricetin, and naringenin) were quantified in the beverage. These compounds are in concordance with the reported data by several studies which previously identified and quantified these compounds in Hibiscus and mint [4,5,10,21].
It has been previously stated that phenolic compounds are associated to astringency and bitter taste in foods and beverages. Hydroxycinnamic acids and their derivatives, as well as flavonol glycosides found in Hibiscus extracts, have been reported to have an astringency taste in red wines [1,22]. This intense flavor is counterbalanced by adding sweeteners, improving consumer preference [1]. Table 2 showed significant differences (p < 0.05) in the total contents of BC prior to the in vitro intestinal digestion. The commercial beverage accounted for 138.05 mg/100 mL, and this total initial content of bioactive compounds was significantly lower (p < 0.05) than the compounds found in the beverage (417.78 mg/100 mL). It must be pointed out that this difference is mainly due to the content of hibiscus acid, which as previously stated, affected consumers acceptance of the product. Moreover, the Hibiscus beverage is free of synthetic additives (i.e., food flavoring or colorants). Thus, their initial bioactive compounds profile found is explained by the mixture of natural ingredients such as Hibiscus and mint. Moreover, the Hibiscus extract used in this preparation comes from specific varieties of Hibiscus sabdariffa L. found in Mexico, which have reported a higher and more diverse content of BC [14]. Borrás-Linares et al. [16] reported that the extracts of PC and flavonoids from Hibiscus could be used for the development of functional food and nutraceuticals. However, the analysis of the bioaccessibility of these compounds is also required [5].

Bioactive Compounds Released during In Vitro Intestinal Digestion
A critical topic around the optimization of the production and formulation of novel functional beverages is the study of the interactions that might occur among the food matrix components (mainly bioactive compounds-fiber-protein-lipids) of the beverage after the mixture of the ingredients used for its production. One of the challenges in the functional beverages industry is to accomplish the "optimal content high enough" of each BC to exert health benefits [23]. Furthermore, it is essential to evaluate the bioaccessibility of the BC to prove beverage functionality effectively. In light of this, several steps have been reported to design a functional beverage, including identifying and quantifying promising bioactive compounds, researching their bioaccessibility, bioavailability, and metabolism, and the potential interactions among the components of the food matrix [22].
Seventeen compounds were identified in the intestinal digestion fraction, with compounds below the limit of quantification (Table 3). A higher content of organic acids and related compounds was observed in the commercial beverage, not in the Hibiscus beverage with significant difference (p > 0.05). After centrifugation of the supernatants on gastrointestinal digestion in the soluble indigestible fraction, only seven compounds were quantified related to the organic acids, all the other BC were not detected, besides the Hibiscus beverage were caffeoylquinic acid and ellagic acid remained in the beverage; however, the content of BC in the Hibiscus beverage was lower than commercial sample, this indicates that most of the compounds quantified before were potentially bioaccesible. In the insoluble indigestible fraction, the commercially beverage the organic acids remain in the residue, indicating that other compounds in this beverage are able to link this compounds and reduce their bioaccessibility. In the case of Hibiscus beverage, the insoluble fraction was lower, this indicates that most of the compounds are bioaccesible (Table 3). Hibiscus acid was the most bioaccessible compound in the Hibiscus beverage, and a decrease in concentration was observed compared with the initial content. Bioaccessibility is also influenced by the decoction process used in beverage preparation, as reported by Mercado-Mercado et al. [24].

Initial Content and Indigestible Fractions after Gastrointestinal Digestion by Groups: Multivariate Data Analysis
PCA was performed on BC concentrations to address patterns among the profiles of BC found in samples at initial content and those non-bioaccessible after gastrointestinal digestion by groups (phenolic acids and flavonoids). For each group, two principal components (PCs) were obtained (Eigenvalues > 1) that explained over 72.7 and 83.3% respectively of the total variance among the samples. PCA analysis showed differentiated BC profile patterns at the different stages evaluated between the beverages. The compounds located in the axis of the PCs at the stage evaluated were based on factors coordinated values <−1.0 for less influence (negative axis) and >1.0 for higher influence (positive axis). Values represent mean ± SD (n = 3). Different lowercase letters in the same row indicate a significant difference (p < 0.05) between beverages for each fraction. (<LOQ) = below the limit of quantification. n.d. = not detected. HB = Hibiscus Beverage. Figures 1a and 2a show a projection of the variables on the factor-plane for the two principal components, showing change in the BC profiles by group. Figure 1a shows the PCA for the group of phenolic acids. It revealed that the differences observed were derived mainly from the influence of compounds found in the initial stage of the hibiscus beverage, corresponding to the PC1 negative axis (ID: 1,3,4,5,7,9,[12][13][14][15][16][17]. PC1 of both groups corroborates Table 3 data because this pattern represents that the Hibiscus beverage has more bioaccessible BC compared to the commercial beverage. PC2 on the positive axis was highly influenced by glycosides such as leucoside, and aglycones such as ellagic acid, and naringenin (ID: 18,25,32). On the other hand, PC1 negative axis comprises compounds that were quantified at relatively low levels (ID: 19,20,22,28,29,30,33,34,35). However, these compounds improve the profile of BC in the hibiscus beverage.
Furthermore, Figures 1b and 2b show that the samples are divided into three welldefined clusters when the profile of BC at the initial stage and intestinal digestion fractions are compared among all the samples. The different clusters showed no correlation between the profile of the bioactive compounds at the initial stage and those bioaccessible since the Hibiscus beverage initial content profile was located in PC1 negative axis. In contrast, its bioaccessible profile changed and was located in both groups' PC1 and PC2 positive axis. However, it must also be remarked that in the commercial beverage, no significant compounds were associated with this group. Thus, the Hibiscus beverage may provide a significant amount of BC potentially bioaccessible. Studies revealed that the profile of BC at the initial stage differs from those released in intestinal digestion [25], confirming the results found in this work.
values <−1.0 for less influence (negative axis) and >1.0 for higher influence (positive axis). Figures 1a and 2a show a projection of the variables on the factor-plane for the two principal components, showing change in the BC profiles by group. Figure 1a shows the PCA for the group of phenolic acids. It revealed that the differences observed were derived mainly from the influence of compounds found in the initial stage of the hibiscus beverage, corresponding to the PC1 negative axis (ID: 1,3,4,5,7,9,[12][13][14][15][16][17]. PC1 of both groups corroborates Table 3 data because this pattern represents that the Hibiscus beverage has more bioaccessible BC compared to the commercial beverage.  PC2 on the positive axis was highly influenced by glycosides such as leucoside, and aglycones such as ellagic acid, and naringenin (ID: 18, 25, 32). On the other hand, PC1 negative axis comprises compounds that were quantified at relatively low levels (ID: 19, 20, 22, 28, 29, 30, 33, 34, 35). However, these compounds improve the profile of BC in the hibiscus beverage.
Furthermore, Figures 1b and 2b show that the samples are divided into three well-

Discussion
After gastrointestinal digestion, the flavonoids and related compounds group found in the indigestible fractions comprise aglycones and glycosidic derivatives. Aglycones such as quercetin and derivatives, such as quercetin-galloylhexoside and Quercetin 3-O-(6-acetil-glucoside), ellagic acid, and myricetin were quantified in the Hibiscus beverage (Table 3). Ellagic acid was recently found as bioaccessible in guava (P. guajava L. 'Amarilla'), this is related to the digestion process, where the enzymes could release ellagic acid from ellagitannin structure linked to the cell wall [26]. Even though ellagic acid may be bioaccessible in the small intestine, its bioavailability is still unclear. Thus it may be more likely to be used as a substrate for the gut microbiota during colonic fermentation [27]. It is suggested that the digestion process (enzymes, pH changes) may increase the release of the bound phenolic compounds and those interacting with the components of the food matrix, mainly dietary fiber [28]. In this context, as reported by Sáyago-Ayerdi et al. [4], phenolic compounds (that interact with dietary fiber or resist the gastrointestinal digestion) from Hibiscus may reach the colon, becoming available substrate for the gut microbiota, which may result as beneficial to the health.
One aspect to consider is that those phenolic compounds released during gastrointestinal digestion may also reach the bloodstream and have health benefits. In this context, the significant reduction in BC found from the initial content in the hibiscus beverage (285.24 mg/100 mL) to the different intestinal fractions (26.70 mg/100 mL) corresponding to a reduction of approximately 90% is probably due to the above-mentioned reason. This behavior is shown in Figure 3.  (Table 3). Ellagic acid was recently found as bioaccessible in guava (P. guajava L. 'Ama-rilla´), this is related to the digestion process, where the enzymes could release ellagic acid from ellagitannin structure linked to the cell wall [26]. Even though ellagic acid may be bioaccessible in the small intestine, its bioavailability is still unclear. Thus it may be more likely to be used as a substrate for the gut microbiota during colonic fermentation [27]. It is suggested that the digestion process (enzymes, pH changes) may increase the release of the bound phenolic compounds and those interacting with the components of the food matrix, mainly dietary fiber [28]. In this context, as reported by Sáyago-Ayerdi et al. [4], phenolic compounds (that interact with dietary fiber or resist the gastrointestinal digestion) from Hibiscus may reach the colon, becoming available substrate for the gut microbiota, which may result as beneficial to the health. One aspect to consider is that those phenolic compounds released during gastrointestinal digestion may also reach the bloodstream and have health benefits. In this context, the significant reduction in BC found from the initial content in the hibiscus beverage (285.24 mg/100 mL) to the different intestinal fractions (26.70 mg/100 mL) corresponding to a reduction of approximately 90% is probably due to the above-mentioned reason. This behavior is shown in Figure 3. In light of this, Rasheed, et al. [29], reported that the BC profile from the initial content of Hibiscus hot and cold beverages was found to have a reasonable correlation for the inhibition of α-glucosidase enzyme, and its regulation is linked to the prevention of type II diabetes. Nevertheless, the BC must be released from the food matrix to present this activity. Figure 4 highlights the possible effects that the compounds found in the Hibiscus beverage (shown in Tables 2 and 3) may exert if they are absorbed into the bloodstream. An important mechanism involved in the anti-inflammatory activities of hibiscus extracts is In light of this, Rasheed, et al. [29], reported that the BC profile from the initial content of Hibiscus hot and cold beverages was found to have a reasonable correlation for the Molecules 2023, 28, 1824 9 of 14 inhibition of α-glucosidase enzyme, and its regulation is linked to the prevention of type II diabetes. Nevertheless, the BC must be released from the food matrix to present this activity. Figure 4 highlights the possible effects that the compounds found in the Hibiscus beverage (shown in Tables 2 and 3) may exert if they are absorbed into the bloodstream. An important mechanism involved in the anti-inflammatory activities of hibiscus extracts is its ability to suppress the generation of oxidative stress and cellular damage in cells. The conjugated forms of quercetin and kaempferol often detected in the plasma after intake of hibiscus could indicate long-lasting cellular antioxidant effects because of their long plasma half-life [30]. Recently, hibiscus acid was reported as a nutraceutical approach for the treatment of chronic myelogenous leukemia due to protein response observed by activation of eIF2α/ATF4 pathway that induced cell cycle arrest at G2/M phase and DNA fragmentation in leukemia K562 cells [31]. There is considerable evidence in the literature showing that BC found in Hibiscus sabdariffa has an antihypertensive effect, which is further supported by a number of in vitro studies demonstrating a vasorelaxant effect of the crude extract of this plant [32]. Hibiscus acid has a direct vasorelaxant effect on the rat aorta, through the inhibition of VDCCs (voltage-dependent calcium channels), inhibiting the influx of extracellular Ca 2+ (a component of the contractile response). Then, this compound may be the constituent responsible for the vascular activity of this plant [33]. It was also found in a recent study that hibiscus acid presents anti-microbial activity against some pathogenic bacteria, concluding that it was one of the compounds responsible for the anti-microbial effect of Hibiscus calyxes [34].
Molecules 2023, 27, x FOR PEER REVIEW 10 of 15 its ability to suppress the generation of oxidative stress and cellular damage in cells. The conjugated forms of quercetin and kaempferol often detected in the plasma after intake of hibiscus could indicate long-lasting cellular antioxidant effects because of their long plasma half-life [30]. Recently, hibiscus acid was reported as a nutraceutical approach for the treatment of chronic myelogenous leukemia due to protein response observed by activation of eIF2α/ATF4 pathway that induced cell cycle arrest at G2/M phase and DNA fragmentation in leukemia K562 cells [31]. There is considerable evidence in the literature showing that BC found in Hibiscus sabdariffa has an antihypertensive effect, which is further supported by a number of in vitro studies demonstrating a vasorelaxant effect of the crude extract of this plant [32]. Hibiscus acid has a direct vasorelaxant effect on the rat aorta, through the inhibition of VDCCs (voltage-dependent calcium channels), inhibiting the influx of extracellular Ca 2+ (a component of the contractile response). Then, this compound may be the constituent responsible for the vascular activity of this plant [33]. It was also found in a recent study that hibiscus acid presents anti-microbial activity against some pathogenic bacteria, concluding that it was one of the compounds responsible for the anti-microbial effect of Hibiscus calyxes [34]. Regarding flavonoids, it has been reported that quercetin-3-sambubioside could be used as a potential antidepressant agent because it promoted the stimulation of the nerve center according to the evaluation of convulsion rate in mice [35]. Related to this, it was also reported that quercetin decreased blood pressure in normotensive and (pre)hypertensive patients showing promising antihypertensive effects [36].  Regarding flavonoids, it has been reported that quercetin-3-sambubioside could be used as a potential antidepressant agent because it promoted the stimulation of the nerve center according to the evaluation of convulsion rate in mice [35]. Related to this, it was also reported that quercetin decreased blood pressure in normotensive and (pre)hypertensive patients showing promising antihypertensive effects [36].

Sample Preparation
About 60 mL of filtered H. sabdariffa beverage was prepared with a mixture of hibiscus varieties cultivated from the region of Jala, Nayarit (21 • 39 15 N, 106 • 32 45 O), Mexico. A decoction with the hibiscus calyces was carried out for 5-6 min at an 80-90 • C temperature, and centrifuged at 700-1100 rpm. The temperature was lowered to 60-70 • C and finally citric acid, mint (added at the beginning of the preparation of the beverage from dried crushed mint leaves), and stevia as a sweetener (97% of purity of rebaudioside A, Metco, Mexico DF, Mexico) were added and all ingredients were homogenized for 5-10 min and cooled to room temperature, according to the patent MX/a/2022/010704. Samples were stored at room temperature (25 • C), and the commercial Hibiscus sabdariffa beverage used as control was purchased from a nutritional store sold as an antioxidant beverage.

Bioaccessibility of Phenolic Compounds after In-Vitro Gastrointestinal Digestion
A Hibiscus beverage and an Hibiscus commercial drink as the control beverage were subjected to a static in vitro gastrointestinal digestion model according to Blancas-Benitez, et al. [37] to evaluate the bioaacessibility of phenolic compounds (PC). Gastric digestion was simulated by adding pepsin (P-7000, Sigma-Aldrich, 0.2 mL of a 300 mg/mL solution in 0.2 M HCl-KCl buffer, pH 1.5, 40 • C, 2 h). Pancreatin (P-1750, Sigma-Aldrich, 3 mL of a 5 mg/mL solution in 0.1 M phosphate buffer, pH 7.5, 37 • C, 2 h) was added in order to simulate the intestinal digestion. After the intestinal digestion, this fraction was used to evaluated the BC released. This method differs from other methods such as the INFOGEST protocol [38], where a dialysis bag is used for the simulation of the passive diffusivity of metabolites in the small intestine; in this study, sample dialysis bags were not used because of the type of sample. The samples from this stage were centrifuged (Hermle Z 323 K; Wehingen, Germany) (3500× g, 15 min, 4 • C), and the supernatant (digested extract) was considered as soluble indigestible fraction. The residue was considered as indigestible-fraction. Both fractions were used to identify the BC by HPLC-DAD-ESI-MS described in the section below.

Determination of Phenolic Compounds and Organic Acids Profile by HPLC-DAD-ESI-MS
An aqueous-organic extraction was performed on the Hibiscus beverage as well as the commercial beverage [39] named as initial stage, and to the supernatant from digested fractions considered as intestinal fraction, soluble indigestible fraction, and residue considered as, insoluble indigestible fraction; all these samples were identified by HPLC-DAD-ESI-MS. These supernatants from aqueous-organic extraction were dispensed in microtubes (Eppendorf, Hamburg, Germany) (2 mL), centrifuged (Vacufuge plus, Eppendorf) (14,000 rpm, 20 min), and filtered through a 0.45 µm nylon membrane filter (Merck Millipore Ltd., Cork, Ireland) and dispensed in chromatographic vials.
The identification of PC by HPLC-DAD-ESI-MS was carried out according to Blancas-Benitez, Pérez-Jiménez, Montalvo-González, González-Aguilar and Sáyago-Ayerdi [38]. An HPLC Agilent 1260 series system (Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent G4212-60008 UV-Vis diode array detector (DAD) and coupled with a 6120 Agilent simple Quadrupole LC/MS with an electrospray ionization interface in negative and positive ionization mode (N 2 as drying gas flow, 13.0 L/min; nebulizer pressure, 40 psi; gas drying temperature, 350 • C; capillary voltage, 3500 V). A volume sample of 10 µL was automatically injected (flow rate 0.4 mL/min) onto a Poroshell 120 EC-C18 column (4.6 mm × 150 mm, particle size 2.7 µm) (Agilent Technologies). The elution gradient was prepared using water containing 0.1 % formic acid as solvent A and acetonitrile as solvent B (Sigma Aldrich). The data analysis was performed using OpenLab CDS, ChemStation Edition software (Agilent Technologies). Characterization of the BC was based on retention time (R T ) in DAD and mass spectrometric signal (single MS scan in the 100-1000 m/z range) directly compared with the R T of analytical standards and also based on previously m/z ions from Hibiscus described in previous studies [4]. External calibration curves were prepared to quantify the compounds identified. When the standard was unavailable, the calibration curve corresponding to its phenolic precursor was used to quantify the compound (Table A1).

Statistical Analysis
All analyses were performed in triplicate; each determination was used to calculate the mean values and standard deviations. Data were analyzed by ANOVA/Fisher's test for all test samples (p < 0.05, n = 3). Datasets of BC profiles were evaluated between samples and intestinal digestion or initial stage using principal components analysis (PCA). Each data set was treated individually, and then compared the patterns displayed by groups of the initial content of CB vs. the content after in vitro digestion of the Hibiscus and the commercial beverage.

Conclusions
Innovation and development of food products with a potential benefit to human health require research related to how bioactive compounds behave in the digestion process, and the data obtained in this work showed that the beverage formulated with H. sabdariffa and mint contains 35 bioactive compounds, in comparison with the 11 from an already market-available product, highlighting the use of Hibiscus in a shot beverage form. The principal component analysis demonstrated that the digestion process significantly affects the bioactive compound profile that may benefit health, distinguishing differentiated groups among the beverages with an increased bioaccessibility of phenolic acids and flavonoids. However, the generation of knowledge related to this process must be carried out comprehensively, and further in vivo studies that evaluate the bioavailability of these bioactive compounds must be taken into account besides the organoleptic and flavor properties when developing a functional food product with such gastronomical importance as H. sabdariffa.